PROJECT SUMMARY Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus. The recent ZIKV outbreak in the Americas uncovered unusual pathologies, were intrauterine infections early during pregnancy were associated with developmental and neurological anomalies such as microcephaly, and the development of Guillain-Barr syndrome in adults. These devastating clinical manifestations together with the rampant spread of the Aedes mosquito make ZIKV a formidable health threat. To date there is no vaccine, and antiviral treatments are elusive. To develop effective therapies however a thorough understanding of the molecular biology of ZIKV is essential. Similar to other flaviviruses, replication of the viral genome likely requires a change in the conformation of the ZIKV genome from a linear to a circular configuration. While putative complementary elements in the ZIKV 5' and 3' untranslated regions (UTRs) have been identified, the mechanism(s) regulating long-range RNA-RNA interactions for circularization are unknown. We have identified two RNA-RNA interactions within the 3' UTR that would either inhibit or promote circularization of the ZIKV genome. In this proposal, I will investigate the role of the dumbbell RNA structure in the 3' UTR in the regulating long-range RNA-RNA interactions between cyclization sequences to direct ZIKV replication. In Aim 1, I will use structural studies and bioinformatics to identify the structure of the 3' dumbbell RNA structures, and molecular biology and virology assays to elucidate the function of the ZIKV 3' dumbbell RNA structures. Using similar approaches in Aim 2, I will confirm putative RNA-RNA interactions in the 3' UTR and investigate how these RNA-RNA interactions regulate the shape of the ZIKV RNA. This proposal will reveal new facets of ZIKV biology, and will uncover the mode by which ZIKV utilizes RNA structure to regulate viral translation and replication. The outcomes of these studies will impact our understanding of the structure, function and regulation of the viral genome of other viruses in the Flaviviridae family such as Dengue virus that presents a growing threat to human health. Moreover, dissecting the pivotal role of RNA structure may eventually provide a foundation for developing new antiviral therapies for this re-emerging and devastating virus.